Phosphorylated compounds readily adsorb to stainless steel surfaces present within the flow path of an HPLC system. This process can lead to poor peak shape, low recovery of sample, and reduction in sensitivity. Here we show that our metal-free column housing technology can improve analysis of these compounds.
HPLC columns are commonly made with stainless steel 316, which is a combination of iron, carbon, manganese, and silicon, with high amounts of nickel, chromium, and molybdenum. The corrosion resistance of stainless steel is caused by the formation of a passive layer of chromium and nickel oxides on the surface, which prevent penetration of corrosive elements. These oxide layers have a high affinity to phosphorylated compounds, particularly those that have accessible phosphate groups.
An illustration of this interaction is shown in Figure 1. As the analyte band travels through the column, it will lose sample through adsorption to the metal surface, resulting in low recovery and a reduction in sensitivity. Adsorption also interferes with uniform sample flow causing peak tailing which can be observed in the resulting chromatogram.
Figure 1: A simplified schematic diagram of analyte loss caused by adsorption of phosphorylated compounds to stainless steel HPLC column housing.
We recently developed metal-free housing to prevent such unwanted interactions. Our design utilizes stainless steel hardware, in order to preserve high pressure capabilities, with internal PEEK coatings (tubing, end fittings, inlet and outlet frits). This removes the largest contribution of metal surfaces within the HPLC sample flow path.
Figure 2 shows several triphosphates under identical conditions in both stainless steel and metal-free housings.
Figure 2: Triphosphates on stainless steel versus metal-free columns.
All triphosphates tested show evidence of adsorption and poor peak shapes with a metal column, which is not seen on our metal-free column. Marked improvements in peak shape and sample recovery, were observed as evidenced by the noticeably larger area under each curve.
When troubleshooting issues with poor peak shape and recovery, contributions of interaction with the column housing are often overlooked. Here we show that there can be considerable interaction of certain analytes with metal surfaces within the column, which can be remedied with the use of a metal-free column. We propose that in any trouble shooting scenario where peak shape cannot be improved through modification of the mobile phase, the use of a metal-free column should be considered.
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